Section of Genetics and Microbiology, Department of Agriculture and Ecology, Faculty of Life Sciences, University of Copenhagen, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark.
Microb Cell Fact. 2010 Jan 22;9:5. doi: 10.1186/1475-2859-9-5.
Chitosanases (EC 3.2.1.132) hydrolyze the polysaccharide chitosan, which is composed of partially acetylated beta-(1,4)-linked glucosamine residues. In nature, chitosanases are produced by a number of Gram-positive and Gram-negative bacteria, as well as by fungi, probably with the primary role of degrading chitosan from fungal and yeast cell walls for carbon metabolism. Chitosanases may also be utilized in eukaryotic cell manipulation for intracellular delivery of molecules formulated with chitosan as well as for transformation of filamentous fungi by temporal modification of the cell wall structures.However, the chitosanases used so far in transformation and transfection experiments show optimal activity at high temperature, which is incompatible with most transfection and transformation protocols. Thus, there is a need for chitosanases, which display activity at lower temperatures.
This paper describes the isolation of a chitosanase-producing, cold-active bacterium affiliated to the genus Janthinobacterium. The 876 bp chitosanase gene from the Janthinobacterium strain was isolated and characterized. The chitosanase was related to the Glycosyl Hydrolase family 46 chitosanases with Streptomyces chitosanase as the closest related (64% amino acid sequence identity). The chitosanase was expressed recombinantly as a periplasmic enzyme in Escherichia coli in amounts about 500 fold greater than in the native Janthinobacterium strain. Determination of temperature and pH optimum showed that the native and the recombinant chitosanase have maximal activity at pH 5-7 and at 45 degrees C, but with 30-70% of the maximum activity at 10 degrees C and 30 degrees C, respectively.
A novel chitosanase enzyme and its corresponding gene was isolated from Janthinobacterium and produced recombinantly in E. coli as a periplasmic enzyme. The Janthinobacterium chitosanase displayed reasonable activity at 10 degrees C to 30 degrees C, temperatures that are preferred in transfection and transformation experiments.
壳聚糖酶（EC 3.2.1.132）水解由部分乙酰化的β-（1,4）-连接的葡萄糖胺残基组成的多糖壳聚糖。在自然界中，壳聚糖酶由许多革兰氏阳性和革兰氏阴性细菌以及真菌产生，其主要作用可能是为了进行碳代谢而从真菌和酵母细胞壁中降解壳聚糖。壳聚糖酶也可用于真核细胞操作，用于将用壳聚糖制成的分子递送到细胞内，以及通过暂时改变细胞壁结构来转化丝状真菌。然而，迄今为止用于转化和转染实验的壳聚糖酶在高温下显示出最佳活性，这与大多数转染和转化方案不兼容。因此，需要具有较低温度下活性的壳聚糖酶。
本文描述了一株产壳聚糖酶的冷活性细菌的分离，该细菌属于 Janthinobacterium 属。从 Janthinobacterium 菌株中分离并鉴定了 876 bp 的壳聚糖酶基因。该壳聚糖酶与糖苷水解酶家族 46 中的壳聚糖酶有关，与链霉菌壳聚糖酶的亲缘关系最近（64%的氨基酸序列同一性）。该壳聚糖酶在大肠杆菌中作为周质酶进行重组表达，其表达量比天然 Janthinobacterium 菌株高约 500 倍。确定温度和 pH 值的最适值表明，天然和重组壳聚糖酶在 pH 值为 5-7 和 45°C 时具有最大活性，但在 10°C 和 30°C 时分别具有 30-70%的最大活性。
从 Janthinobacterium 中分离出一种新型的壳聚糖酶及其相应基因，并在大肠杆菌中作为周质酶进行重组表达。Janthinobacterium 壳聚糖酶在 10°C 到 30°C 的温度下具有合理的活性，这是转染和转化实验中首选的温度。
